Voltage-dependent anion channel (VDAC; mitochondrial porin) is found in the outer mitochondrial membrane in all eukaryotic cells. VDAC is a central player in cell energy metabolism and have a key role in mitochondria-mediated apoptosis, and controls the fluxes of ions and metabolites between the mitochondrion and the cytosol. VDAC also provides a point of convergence for a variety of cell survival and death signals, mediated via its association with various ligands and proteins. VDAC is a key player in mitochondria-mediated apoptosis, participating in the release of mitochondria pro-apoptotic proteins (e.g. cytochrome c, apoptosis inducing factor (AIF) and second mitochondria-derived activator of caspases (Smac/DIABLO)) to the cytosol and interacts with apoptosis regulatory proteins such as Bcl-2, Bcl-xL and hexokinase (HK).
Three mammalian isoforms of VDAC are known, VDAC1, VDAC2 and VDAC3, where VDAC1 is the major isoform expressed in mammalian cells (De Pinto, V., et. al. 2010. Biochim Biophys Acta 1797:1268-1275). Blachly-Dysion et al. (Blachly-Dyson E et al., 1993. J Biol Chem. 268(3):1835-41) disclosed the cloning and functional expression in yeast of two human VDAC isoforms, VDAC1 and VDAC2. U.S. Pat. No. 5,780,235 discloses two VDAC sequences, which were named HACH (Human Voltage-Dependent Anion Channel), subsequently identified as VDAC2 and VDAC3. That patent provides genetically engineered expression vectors, host cells containing the vector, a method for producing HACH and a method for identifying pharmaceutical compositions inhibiting the expression and activity of HACH as well as the use of such compositions for the treatment of cancer and proliferative diseases.
Apoptosis, also known as programmed cell death, plays a central role in, inter alia, development, immune cell regulation and tissue homeostasis in multicellular organisms. Genetic and molecular analysis from various species has indicated that the apoptotic pathway is highly conserved. In addition to being essential for normal development and maintenance, apoptosis is important in the defense against viral infection and in preventing cancer. Mitochondria play an important role in apoptotic cell death. The release of apoptogenic proteins such as cytochrome c from the mitochondrion intermembranal space into the cytoplasm of the cell initiates a cascade of steps involved in caspase activation that executes the cell death program. Substantial evidence links VDAC1 to apoptosis and suggests that VDAC1 is a critical player in the release of apoptogenic proteins from mitochondria in mammalian cells (Lemasters, J. J., and Holmuhamedov, E. 2006. Biochim. Biophys Acta 1762:181-190; Shoshan-Barmatz V et al. 2010. Molecular Aspects of Medicine 31(3):227-286; Shoshan-Barmatz V and Ben-Hail D. 2012. Mitochondrion 12(1):24-34; Shoshan-Barmatz V and Golan M. 2012. Current Medicinal Chemistry 19(5):714-35; Shoshan-Barmatz, V et al. 2015. Biochim Biophys Acta, 1848(10 Pt B):2547-75).
It has been previously shown that peptides derived from VDAC are capable of inducing apoptosis (Prezma T et al. 2013. Cell Death and Disease 4:e809).
U.S. Pat. Nos. 8,119,601 and 8,648,045 to the inventor of the present invention and others disclose isolated VDAC1 derived peptides that are capable of inducing apoptosis in a cell and to pharmaceutical compositions comprising same useful in the treatment of disease associated with aberrant apoptosis, particularly cancer. The peptides are derived from the N-terminal domain of VDAC1 as well as from VDAC1 β-strand 14 and its cytosolic β-loop.
International Patent Application Publication No. WO 2015/011711 the inventor of the present invention and others discloses short peptides based on the amino acids sequence of the N-terminal domain of the human mitochondrial protein voltage-dependent anion channel 1 (VDAC) and to peptide conjugates further comprising a cell permeability enhancing moiety. The peptides, peptide conjugates and pharmaceutical composition comprising same are useful for treating diseases characterized by cell hyper-proliferation or resistance to cell death, particularly cancer.
Prezma et al. (Prezma T et al., 2013. Cell Death Dis. 19(4):e809) showed that VDAC1-based peptides, including short peptides, selectively induced cells death of peripheral mononuclear cells (PBMC) from patients with B-chronic lymphocytic leukemia (CLL) while exhibiting minor effects on PBMCs from healthy donor.
Non-alcoholic fatty liver disease (NAFLD) is often “silent” liver disease and characterized by an excessive abnormal accumulation of fatty acids and triglycerides within the cytoplasm of the hepatocytes and other liver cells of non-alcohol users. NAFLD has emerged as a significant public health concerns in Western societies and is the most common cause of abnormal liver function. NAFLD is a continuum chronic fatty liver diseases ranging from benign hepatic steatosis and its progressive form called non-alcoholic steatohepatitis (NASH). In NASH, fat accumulation in the liver is associated with inflammation and different degrees of scarring. NASH is a potentially serious condition that carries a substantial risk of progression to end-stage liver disease, cirrhosis and hepatocellular carcinoma (HCC). Some patients who develop cirrhosis are at risk of liver failure and may eventually require a liver transplant.
Nonalcoholic fatty liver disease is classified as isolated fatty liver (IFL) and NASH. In both IFL and NASH there is an abnormal amount of fat in the liver cells, but, in addition, in NASH there is inflammation within the liver, and, as a result, the liver cells are damaged, and when die, they are replaced by scar tissue leading to cirrhosis.
The cause NAFLDs is complex and not completely understood. With that, NAFLD is universally considered as the hepatic manifestation of the metabolic syndrome (MS) and insulin resistance is regarded as its key pathophysiological hallmark. Given the strong association of NAFLD with metabolic syndrome as well as the worldwide epidemic of obesity, the prevalence of NAFLD and NASH are increasing. NAFLD is more prevalent in cohorts of patients with pre-existing metabolic conditions than the general population. Specifically, type II diabetes mellitus and NAFLD have a particularly close relationship. A study of patients with type II diabetes mellitus reported a 69% prevalence of ultrasonographic NAFLD. However, no relationship was evident between diabetic degenerative complications or glycaemic control and the presence of NAFLD. The prevalence of simple steatosis in obese individuals ranges from 30% to 37%, while in NAFLD ranges from 57% of overweight individuals attending out-patient clinics to 98% of nondiabetic obese patients.
There are no drugs currently approved to prevent or treat NAFLD or NASH and thus current therapies rely on lifestyle modifications and treatments with disease associated with NAFLD. The most important recommendations given to persons with this disease include reducing weight (if obese or overweight), following a balanced and healthy diet, increasing physical activity, avoiding alcohol and unnecessary medications, and in some cases bariatric surgery, where the main goal is to reduce disease symptoms after diagnosis.
Mitochondria can influence cell fate at the levels of energy production, lipid metabolism, production, and detoxification of reactive oxygen species (ROS) and release of pro-apoptotic proteins. Various studies showed that mitochondria harbor prominent morphologic role of these organelles in the pathogenesis of hepatosteatosis.
A major obstacle to the in vivo therapeutic use of peptides is their susceptibility to proteolytic degradation. Retro-inverso peptides are peptides whose amino acid sequence is reversed and the α-center chirality of the amino acid subunits is inverted as well. Usually, these types of peptides are designed by including D-amino acids in the reverse sequence to help maintain side chain topology similar to that of the original L-amino acid peptide and make them more resistant to proteolytic degradation. Other reported synonyms for these peptides in the scientific literature include All-D-Retro Peptides, Retro-Enantio Peptides, Retro-Inverso Analogs, Retro-Inverso Analogues, Retro-Inverso Derivatives, Retro-Inverso peptides and Retro-Inverso Isomers. D-amino acids represent conformational mirror images of natural L-amino acids occurring in natural proteins present in biological systems. There are also partially modified retro-inverso analogues of linear peptides in which only some of the peptide bonds are reversed and the chirality of the amino acid residues in the reversed portion is inverted.
Peptides that contain D-amino acids are typically less susceptible to proteolytic degradation and have a longer effective time when used as pharmaceuticals. If properly designed, retro-inverso peptides can have binding characteristics similar to L-peptides. Retro-inverso peptides are useful candidates for the study of protein-protein interactions by designing peptidomimetics that mimic the shape of peptide epitopes, protein-protein, or protein-peptide interfaces. Retro-inverso-peptides are attractive alternatives to L-peptides used as pharmaceuticals. These peptides have been reported to elicit lower immunogenic responses compared to L-peptides. However, although retro-inverso analogues exhibit increased metabolic stability, their biological activity is often greatly compromised (Guichard G et al., 1994. Proc. Natl. Acad. Sci. U.S.A., 91:9765-9769).
There is an unmet need for and it would be highly advantageous to have VDAC1-based peptides exhibiting improved pharmacokinetic characteristics while at least retaining their biological activity.